Human monoclonal antibodies directed against the transmembrane glycoprotein (gp41) of human immunodeficiency virus-1 (HIV-1)

ABSTRACT

Disclosed is a human monoclonal antibody produced by the hybridoma designated Clone 3 and having A.T.C.C. Accession No. CRL 10198. The Clone 3 human monoclonal antibody immunologically binds to a conserved epitope on the transmembrane envelope glycoprotein gp41 of Human Immunodeficiency Virus Type 1 (HIV-1) having the amino acid sequence GCSGKLIC.

This application is a continuation-in-part of U.S. application Ser. No.07/396,751, filed Aug. 24, 1989, now pending.

FIELD OF THE INVENTION

This invention relates to anti-HIV-1 monoclonal antibodies andspecifically to monoclonal antibodies which bind to a viral epitope,thereby neutralizing the virus. The invention also relates to continuouscell lines capable of producing the antibodies and to the peptidesrecognizable by the antibodies. The antibodies and antigens of thisinvention are useful for diagnosis, prognosis, prophylaxis and therapy.This invention also relates to prognostic tests for viral diseases, andparticularly prognostic tests for Acquired Immunodeficiency Syndrome(AIDS).

BACKGROUND OF THE INVENTION

The human immunodeficiency virus (HIV-1) has been established as theprimary etiologic agent in the pathogenesis of acquired immunodeficiencysyndrome (AIDS) and related disorders. (Barre-Sinoussi, et al. Science(1983) 220:868-871; Gallo, et al., Science (1984) 224:500-503; Levy, etal., Science (1984) 225:840-842).

The CD4+ cells play a central role in HIV infection. (Fauci, Science(1988) 239:617-622). CD4 is a molecule present on the surface of certainlymphocytes and, to a lesser degree, macrophages. The CD4 molecule playsa significant role in the function of T4 helper lymphocytes and servesas a marker for such cells. (Gallo, R. C. and Montagnier, L., ScientificAmerican (1988) 259:41-48.) The virus uses the CD4 receptor to gainentry into a number of cells. (Dalgleish, et al., Nature (1984)312:763-767). The envelope glycoprotein, gp160, is the precursor to thegp120, which specifically binds to the surface receptor (CD4) of CD4+cells, and the gp41, the transmembrane (TM) glycoprotein which initiatescell-membrane fusion, leading to the formation of multinucleated giantcells commonly called syncytia. (Kowalski, Science (1987)237:1351-1355). Fusion leads to the death of the syncytial cells. WhileHIV-1 may also cause cell death through mechanisms independent of cellfusion, data suggest that the formation of syncytia contributes to theprogressive depletion of CD4+ cells (T4 helper lymphocytes),quantitatively and functionally. (Lifson, et al., Nature (1986)323:725-728). This is the most profound hematologic feature and hallmarkassociated with acquired immunodeficiency syndrome (AIDS) (Broder, S. M.and Gallo, R. C., N. Eng. J. Med. (1984) 311:1292-1297), as demonstratedby impaired cell-mediated immunity.

Infection of humans with HIV-1 leads to a humoral immune response by Blymphocytes resulting in the production of antibodies directed againstmost of the viral structural antigens. A particular subset of antibodiesis directed against HIV envelope antigens (gp120 and gp41) which may beinvolved in induction of active immunity. (Matthews, et al., AIDSResearch and Human Retroviruses (1987) 3:197-206). Neutralization assayswith sera from HIV-infected individuals (Robert-Guroff, et al., Nature(1985) 316:72-74; Rasheed, et al., Virology (1986) 150:1-9) or fromimmunized animals, suggest that the envelope glycoprotein containsepitope(s) that elicit antibodies capable of neutralizing HIV infectionin vitro. As an in vivo corollary, it has been demonstrated that highneutralizing antibody titers correlated with a better clinical outcome,and low or decreasing neutralizing antibody titer signaled poorprognosis. (Robert-Guroff, et al., AIDS Research and Human Retroviruses(1988) 4:343-350). A decrease in average antibody titers has beenclinically observed in late stages of infection, particularly withregard to antibodies directed against the HIV envelope epitopes andspecifically against the TM gp41 region containing the amino acidsequence against which the herein described human monoclonal antibody isbiologically reactive. (Shafferman, et al., AIDS Research and HumanRetroviruses (1989) 5:33-39; Chiodi, et al., J. Med. Virol. (1987)23:1-9; McPhee, et al., FEBS Lett. (1988) 233:393-396).

Measures capable of boosting the neutralizing antibody titer ofindividuals already infected with the virus, eliciting high-titerneutralizing antibodies (i.e., active immunotherapy), or increasingneutralizing antibodies (i.e., passive immunotherapy) in individuals atrisk would prove beneficial in controlling viral spread in vivo or inpreventing new infection. (Robert-Guroff, et al., AIDS Research andHuman Retroviruses (1988) 3:343-350).

The present invention makes possible the measures cited above. Anyattempts at passive immunotherapy will require the production of largequantities of antibody on a routine basis. The development of acontinuous cell line accommodates this. The monoclonality of theantibody enables the administration of reactive physiological amounts ofthe antibody since all of the antibody being administered is directedagainst the biologically active epitope of the virus, unlike polyclonalserums which contain antibodies against other structural proteins aswell. The potential immunogenicity of the peptide sequence of thisepitope, as evidenced by the immunogenicity of a peptide of similarsequence, will enable safe and effective vaccination of individuals,thereby avoiding the great risks involved in immunizing with attenuatedor even killed viruses.

The present invention is further directed to a kit and a method fordetecting the presence and determining concentration of an antibody thatinhibits HIV-1 fusion-associated epitope, a peptide on gp41 with theamino acid sequence represented by GCSGKLIC (SEQ NO:1). The detectionand quantitation method includes an enzyme-linked immunosorbent assay(ELISA).

The determination of the concentration of the antibody that inhibitsHIV-1 fusion-associated epitope on gp41, present in body fluids of apatient seropositive for HIV-1 antibodies, provides the physician withan objective means to form a prognosis for each individual case andassists the clinician therefore in determining the appropriateness toinitiate medical intervention or change therapy.

SUMMARY OF THE INVENTION

The present invention is concerned with a novel human monoclonalantibody which defines and neutralizes a biologically functionalantigenic/immunogenic site on the HIV-1 transmembrane (TM) envelopeglycoprotein.

The invention of the human monoclonal antibody, the production of theantibody, the identification of the epitope (peptide) to which theantibody binds, and the delineation of the biologically importantfunction of the defined epitope, are achieved and described in detail bythe following outlined immunochemical methods and biological assays.

The antigenic/immunogenic peptide identified as the epitope (targetantigen) is contained within the twelve amino acid (L-form) sequence:leucine-glycine-isoleucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine(SEQ. No. 2). Since antigenic determinants have been reported to berepresented by as few as five amino acid residues, the actual epitopemay be a truncation of this sequence. (Barrett, J. T., Textbook ofImmunology (1983) p. 41). Specifically, the human monoclonal antibodyimmunochemically binds to a conserved peptide of the HIV-1 transmembrane(TM) glycoprotein designated gp41, and, as a consequence of thisantibody-antigen reaction, biologically blocks syncytia formationbetween HIV-1 virally infected human lymphocytes and uninfectedlymphocytes (CD4+ cells).

The described human monoclonal antibody binds to the identified viralepitope, specificallyleucine-glycine-isoleucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine,and the fusion of HIV-1-infected CD4+ cells with non-infected CD4+ cellsis thereby blocked. Cell fusion is a biologically functional processthat can therefore be ascribed to this conserved immunodominant region.

The human monoclonal antibody described (Anti-gp41) inhibits fusion ofCD4+ cells caused by HIV-1 and therefore blocks infection via binding tothe gp41 transmembrane glycoprotein, specifically the fusion-associatedepitope as delineated. This inhibition signifies the utility ofAnti-gp41 in passive immunotherapy. Other fusion-inhibiting agents, suchas synthetic inhibitory peptides, are also disclosed. Administration ofthe retrovirus/vaccine containing the immunogenic fusion-associatedepitope/subunit will elicit fusion blocking antibody (as evidenced bythe human B cell line identified from the HIV-1-infected, naturallyimmunized, patient/donor), thereby providing protection against theinfection and depletion of CD4+ cells and, therefore, preventing thedevelopment of acquired immunodeficiency syndrome. This protectionevidences the utility of gp41, or an appropriate subunit thereof, inactive immunotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention and forfurther advantages thereof, reference is made to the following DetailedDescription taken in conjunction with the accompanying Drawings, inwhich:

FIG. 1 is a depiction of the Western blot described in Example 3;

FIG. 2 is a graph of the results of the syncytium-forming microassaydescribed in Example 11;

FIG. 3 presents the 23 amino acid peptide sequences for SIV, used in theELISA by Johnson et al., as reported in AIDS Research and HumanRetroviruses (1988) 4:159-164, and the analogous regions for HIV-2 andHIV-1;

FIG. 4 illustrates a linear comparison of the peptide sequences testedin determining the specificity of Anti-gp41;

FIG. 5 presents the linear representation of amino acid sequences (threeletter abbreviation) contained within the transmembrane glycoproteingp41 of HIV-1 for Peptide 2 and Peptide 6120;

FIG. 6 illustrates the fine epitope mapping for Peptides 232-240;

FIG. 7 presents the linear representation of amino acid sequences (threeletter abbreviation) contained within the transmembrane glycoproteingp41 of HIV-1 for Peptide 237 and Peptide 238;

FIG. 8 shows the reduced and non-reduced Western Blot for the donorpatient plasma and for Clone 3 antibody; and

FIG. 9 illustrates immunofluorescence profiles by flow cytometry forHIV-infected Sup-T1 cells and uninfected Sup-T1 cells.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention is to novel human monoclonal antibodies that arebiologically reactive against a specific immunodominant peptide regionwithin the transmembrane glycoprotein gp41 envelope component of thehuman immunodeficiency virus (HIV-1), delineation of the peptide region(epitope) to which the human monoclonal antibody binds, and immunologicdescription of the associated biological function of the epitope. Theinvention also encompasses the specific immunologically reactive andbiologically functional analogous peptide regions (immunogens) on HIV-2and SIV to elicit the production of corresponding neutralizingantibodies against HIV-2 in humans, and SIV in non-human primates.

The following describes the creation of cells which are capable ofproducing, in Vitro, ad infinitum, human monoclonal antibodies that arebiologically active against the transmembrane glycoprotein gp41 ofHIV-1.

Based on the fact that mononuclear cells circulating in the peripheralblood of HIV-1-infected individuals produce antibodies against HIVantigens, an asymptomatic patient who had high titers of plasmaantibodies against HIV was selected as a donor of B-lymphocytes. TheB-lymphocytes collected from this patient were subsequently transformedby Epstein-Barr Virus (EBV). Seven anti-HIV antibody-producing humanlymphoblastoid cell lines have been obtained in this manner and havebeen stable with respect to antibody production. In order to furtherassure stability and augment antibody production, an aliquot of each ofthe created EBV transformed human cell lines has been fused with aheteromyeloma (mouse-human hybrid myeloma) as a fusion partner toproduce a hybridoma counterpart.

Supernatants from the resultant hybridomas were screened for antibodiesagainst HIV. Cell lines testing positive for anti-HIV antibodyproduction were subcloned twice and secreted antibody was then testedspecifically for binding against the envelope glycoproteins of the virus(i.e., gp41 and gp120). One of seven human monoclonal antibodies testingpositive for anti-gp41 reactivity (designated Clone 3 antibody) has beenpurified from the cell culture supernatants by affinity chromatographictechniques. This human monoclonal antibody (not an animal derivedmonoclonal antibody) has been demonstrated to have biological activityat physiologic concentrations against HIV-1 (specifically HTLV-IIIB) ina fusion (syncytium) inhibition, in vitro assay; and in neutralizationof free HIV-1 (specifically SF2) infectivity of human mononuclear cellsin culture. The specific level of fusion inhibition and inhibition offree virus infectivity achieved is detailed in Example 11 and Example18, respectively, discussed hereinafter.

Development of Clone 3 cell line is detailed further in Example 1discussed hereinafter. Clone 3 was deposited with the American TypeCulture Collection (ATCC) in Rockville, Md. on Aug. 10, 1989 andreceived accession number ATCC CRL 10198.

Additionally, the specific amino acid sequence (epitope) to which thehuman monoclonal antibody binds has been ascertained, thereby ascribingthe associated biological function of cell fusion to the epitope(fusion-associated epitope). Epitope mapping is discussed hereinafter inExample 8, Example 9, and Example 10.

Other investigators have utilized knowledge of antigenic epitopes todevelop "complementary" peptides that are capable of inhibitingepitope-epitope (prototopes) binding or inhibiting the binding of anenzyme to its substrate. (McPhee, et al., Cold Spring Harbor Symposium(1988) p. 17; Lambert, et al., V International Conference on AIDS(1989), Abstract No. W.C.O.11, p. 526). In either instance, developmentof complementary peptides is facilitated since a complementary peptidealready exists and has been tested in nature. Such peptides were foundto inhibit production of the mature viral proteins reverse transcriptaseand p24, or to inhibit or delay syncytia formation.

Similarly, a complementary peptide could be synthesized which would becapable of binding to the gp41 fusion-associated epitope disclosed,thereby preventing fusion between HIV-1-infected and uninfected cells.The synthetic peptide could then be administered therapeutically.

The biological reactivity of the monoclonal antibody and the biologicalfunction of the epitope to which it binds reveal the invention's utilityfor passive and active therapeutic intervention in the treatment ofacquired immunodeficiency syndrome. Specifically, data to support theefficacy of passive immunotherapy in chimpanzees have been published,for it has been determined that neutralization of in vivo HIV-1infectivity can be mediated by in vitro neutralizing antibody directedagainst the gp120 major, yet hypervariable, neutralizing epitope.(Emini, et al., V. International Conference on AIDS (1989), Abstract No.Th.C.O.30, p. 538). The human monoclonal antibody can be administered topatients who lack neutralizing antibodies against this epitope withingp41, thereby providing passive immunotherapy. In a parallel humanstudy, data from recent clinical trials (Jackson, et al., Lancet (1988)2:647-652; Karpas, A., Proc. Natl. Acad. of sciences (U.S.A.) (1988)85:9234-9237) have demonstrated that passive immunization improved thestatus of patients with advanced AIDS. In those trials, passiveimmunization was accomplished by transfusing plasmas containingantibodies from asymptomatic AIDS patients into the symptomatic AIDSrecipients.

In a similar approach, another therapeutic use of the monoclonalantibody of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the fusion-associated epitope could elicit anactive anti-gp41 response. (Linthicum, D. S. and Farid, N. R.,Anti-Idiotypes, Receptors, and Molecular Mimicry (1988), pp. 1-5 and285-300).

Likewise, active immunization can be induced by administering theantigenic and immunogenic immunodominant epitope as a component of asubunit vaccine. Vaccination could be performed orally or parenterallyin amounts sufficient to enable the recipient to generate protectivefusion inhibiting (neutralizing) antibodies against this biologicallyfunctional region, prophylactically or therapeutically. Additionally,the peptideleucine-glycine-leucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine(SEQ NO:28), or a truncated version thereof, may be used in place of thenative peptide sequence disclosed as the fusion-associated epitope,since this peptide has been shown to be capable of equivalently bindingpolyclonal antibodies which recognize the native epitope containing anisoleucine instead of a leucine at the third amino acid position in thesequence above. (Gnann, et al., Science (1987) 237:1346-1349).

The twelve amino acid residue peptide which is immunologically reactivewith the HIV-1 specific fusion blocking human monoclonal antibody,described immunochemically and biologically in the text, is useful incompositions of subunit vaccines to elicit the production of protectivefusion blocking antibodies against HIV-1 in animals including man. Thehost can be actively immunized with the antigenic/immunogenic peptide inpure form, a fragment of the peptide, or a modified form of the peptide.

One or more amino acids, not corresponding to the original proteinsequence, can be added to the amino or carboxyl terminus of the original12-mer (i.e., 12 amino acid peptide), or truncated 11-mer, 10-mer,9-mer, 8-mer, or even 7-mer peptides. Such extra amino acids are usefulfor coupling the peptide to another peptide, to a large carrier protein,or to a support. Amino acids that are useful for these purposes include:tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivativesthereof.

Alternative protein modification techniques may be used, e.g., NH₂-acetylation or COOH-terminal amidation, to provide additional means forcoupling the peptide to another protein or peptide molecule or to asupport.

The novel peptide sequence is set forth below in a general formulacommon to HIV-1, HIV-2, and SIV:

    X-a-b-c-tryptophan-glycine-cysteine-x-x-x-x-x-cysteine-Y-Z.

The specific novel peptide sequence, and truncated sequences, for eachof the comparable analogous conserved immunodominant regions from HIV-1,HIV-2, and SIV are set forth in the following formulae.

For the Human Immunodeficiency Virus-1 (HIV-1), a conservedimmunodominant, antigenic/immunogenic twelve amino acid residue peptide(amino acids numbers 598-609, Gnann numbering system, id.), a structuralcomponent of the transmembrane glycoprotein gp41, is immunologicallyreactive with the HIV-1 specific fusion blocking human monoclonalantibody. The novel peptide sequence and the truncated sequences are setforth in the formula below:

    X-a.sub.1 -b.sub.1 -c.sub.1 -tryptophan-glycine-cysteine-.sup.1 x.sub.1 -.sup.2 x.sub.1 -.sup.3 x.sub.1 -.sup.4 x.sub.1 -.sup.5 x.sub.1 -cysteine-Y-Z

where X is either a H of the amino terminal NH₂ group of the peptide oran additional amino acid bonded to the amino terminal NH₂ group of thepeptide, the additional amino acid being selected to facilitate couplingof the peptide to a carrier protein; Y is absent or cysteine; and Z isOH or NH₂, and

a₁ is leucine

b₁ is glycine

c₁ is isoleucine

¹ x₁ is serine

² x₁ is glycine

³ x₁ is glycine

⁴ x₁ is leucine

⁵ x₁ is isoleucine

Alternatively, a truncated peptide can be produced where a is present,with b and c also being present, to represent the original 12-mer; a isabsent, with b and c being present, to represent an 11-mer; a and b areabsent, with c only being present, to represent a 10-mer; a and b and care absent, to represent a 9-mer, depicted by the following sequenceformula: tryptophan-glycine-cysteine-x-x-x-x-x-cysteine which is commonto analogous positions within the three comparable epitopes for HIV-1,HIV-2 and SIV. Similarly, the tryptophan and glycine residues may alsobe deleted resulting in the production of an 8-mer and 7-mer,respectively.

For the Human Immunodeficiency Virus-2 (HIV-2), a conservedimmunodominant, antigenic/immunogenic twelve amino acid residue peptide(amino acids numbers 592-603, Franchini numbering system as reported byJohnson, et al., AIDS Research and Human Retroviruses (1988) 4:159-164)which is a structural component of the transmembrane glycoprotein, andthe truncated sequences are set forth in the formula below:

    X-a.sub.2 -b.sub.2 -c.sub.2 -tryptophan-glycine-cysteine-.sup.1 x.sub.2 -.sup.2 x.sub.2 -.sup.3 x.sub.2 -.sup.4 x.sub.2 -.sup.5 x.sub.2 -cysteine-Y-Z

where X is either a H of the amino terminal NH₂ group of the peptide oran additional amino acid bonded to the amino terminal NH₂ group of thepeptide, the additional amino acid being selected to facilitate couplingof the peptide to a carrier protein; Y is absent or cysteine; and Z isOH or NH₂, and

a₂ is leucine

b₂ is asparagine

c₂ is serine

¹ x₂ is alanine

² x₂ is phenylalanine

³ x₂ is arginine

⁴ x₂ is glutamine

⁵ x₂ is valine

Alternatively, a truncated peptide can be produced where a is present,with b and c also being present, to represent the original 12-mer; a isabsent, with b and c being present, to represent an 11-mer; a and b areabsent, with c only being present, to represent a 10-mer; a and b and care absent, to represent a 9-mer as described above. The tryptophan andglycine residues may also be deleted resulting in the production of an8-mer and 7-mer, respectively.

For the simian immunodeficiency virus (SIV), a conserved immunodominant,antigenic/immunogenic twelve amino acid residue peptide (amino acidsnumbers 617-628, Franchini numbering system, id.), which is a structuralcomponent of the transmembrane glycoprotein gp32, and the truncatedsequences are set forth in the formula below:

    X-a.sub.s -b.sub.s -c.sub.s -tryptophan-glycine-cysteine-.sup.1 x.sub.s -.sup.2 x.sub.s -.sup.3 x.sub.s -.sup.4 x.sub.s -.sup.5 x.sub.s -cysteine-Y-Z

where X is either a H of the amino terminal NH₂ group of the peptide oran additional amino acid bonded to the amino terminal NH₂ group of thepeptide, the additional amino acid being selected to facilitate couplingof the peptide to a carrier protein; Y is absent or cysteine; and Z isOH or NH₂, and

a_(s) is leucine

b_(s) is asparagine

c_(s) is alanine

¹ x_(s) is alanine

² x_(s) is phenylalanine

³ x_(s) is arginine

⁴ x_(s) is glutamine

⁵ x_(s) is valine

Alternatively, a truncated peptide can be produced where a is present,with b and c also being present, to represent the original 12-mer; a isabsent, with b and c being present, to represent an 11-mer; a and b areabsent, with c only being present, to represent a 10-mer; a and b and care absent, to represent a 9-mer as described above. The tryptophan andglycine residues may also be deleted resulting in the production of an8-mer and 7-mer, respectively.

The three-letter and single-letter abbreviations for the amino acids areas follows:

Ala(A), alanine

Arg(R), arginine

Asn(N), asparagine

Asp(D), aspartic acid

Cys(C), cysteine

Gln(Q), glutamine

Glu(E), glutamic acid

Gly(G), glycine

His(H), histidine

Ile(I), isoleucine

Leu(L), leucine

Lys(K), lysine

Met(M), methionine

Phe(F), phenylalanine

Pro(P), proline

Ser(S), serine

Thr(T), threonine

Trp(W), tryptophan

Tyr(Y), tyrosine

Val(V), valine

Description of Non-Human Primate and Human Clinical Subunit VaccineTrials

The only animal that can be reproducibly infected with HIV, thusproviding an experimental system for testing the effectiveness ofprototype vaccines, is the chimpanzee. Although chimpanzees can beexperimentally infected with HIV-1, clinical disease has not, to date,developed in infected animals. Furthermore, the supply of chimpanzeesavailable for biomedical research is limited since the assignation ofthe chimp as an endangered species. The recently described simianimmunodeficiency virus (SIV, STLV-III), Desrosiers, R. C. and Letvin, N.L., Rev. Infect. Dis. (1987) 9:438-446, provides a potentially moreuseful model system based on the infection of rhesus macaques andAfrican green monkeys (Kanki, et al., Science (1985) 230:951-1954).

SIV, although closely related to HIV-1 (Hirsch, et al., Cell (1987)49:307-319; Franchini, et al., Nature (1987) 328:539-543; Chakrabarti,et al., Nature (1987) 328:543-547), is genetically more related to HIV-2(Franchini, et al., supra, Chakrabarti, et al., supra), which alsocauses human AIDS (Clavel, et al., N. Eng. J. Med. (1987)316:1180-1185). And, most importantly, SIV induces clinical AIDS similarto the human syndrome in infected macaques. (Desrosiers, R. C. andLetvin, N. L., supra).

The serologic diagnosis of SIV infection in monkeys has been made usingtraditional antibody assays, including enzyme-linked immunosorbent assay(ELISA) with whole-virus lysate as antigen. For the serologic diagnosisof HIV-1 infection in humans, a more sensitive, specific, as well assimple, diagnostic method has been investigated in ELISA systems usingsynthetic peptides as solid-phase antigens (site-directed ELISA). Inparticular, selected synthetic peptides that correspond to sequencesfrom the amino-terminal half (amino acids 586-620) of the transmembraneglycoprotein (gp41) have reacted with over 99% of sera from human AIDSpatients (Wang, et al., Proc. Natl. Acad. Sci. (U.S.A.) (1986)83:6159-6163; Smith, et al., J. Clin. Microbiol. (1987) 25:1498-1504;Gnann, et al., Science (1987) 237:1346-1349; Gnann, et al., J. Infect.Dis (1987) 156:261-267; Chiodi, et al., J. Med. Virol. (1987) 23:1-9).

A synthetic peptide from an analogous region of the SIV transmembraneglycoprotein (gp32) is highly immunoreactive with sera from SIV-infectedprimates. This reactivity extends across four primate species from threegenera and indicates infection by at least two distinct isolates of SIVin experimentally and naturally infected monkeys. (Johnson, et al., AIDSResearch and Human Retroviruses (1988) 4:159-164.) Preliminaryexperiments also demonstrated that this peptide from the SIV gp32reacted strongly with sera from humans infected with HIV-related WestAfrican viruses HIV-2 (HTLV-IV). Furthermore, reactivity with thispeptide was specific for infection with the West African viruses, sincethese same sera did not react with the analogous peptides from HIV-1.(Norrby, et al., Nature (1987) 329:248-250.)

Sequences of the synthetic peptide used in the ELISA to detectantibodies against the SIV transmembrane glycoprotein (gp32) and theanalogous regions from HIV-2 and HIV-1 are presented in FIG. 3, usingthe single-letter abbreviations for the amino acids. The sequences arenumbered according to the Johnson reference, supra. It should be notedthat the sequence for HIV-1 contains the 12 amino acid sequence ofpeptide 2 (amino acid residues 593-604 in the Franchini numberingsystem) described hereinafter in Example 8 and used in epitope mappingto determine the specificity for the human monoclonal antibody Anti-gp41produced by Clone 3.

Non-identical amino acid residues for HIV-1 and HIV-2 compared with theanalogous regions with the SIV sequence are denoted by doubleunderlining. The two cysteine amino acid residues are marked by a singlyunderlined C.

The three analogous peptide sequences delineated above, consisting of 23amino acid residues, have some unique biochemical structural featuresthat are common to each. Two closely spaced cysteine residues may serveto orient the peptide in a similar configuration in order that anessential biological function can proceed, perhaps via disulfidebonding. The importance of these cysteine residues for maintainingantigenicity has been demonstrated recently for a similar HIV-1 peptide.

Sequential single amino acid deletions from the amino terminus of the 12amino-acid peptide (amino acid residues 598-609 in the Gnann numberingsystem, which corresponds to amino acid residues 593-604 in theFranchini numbering system) of gp41 revealed a minor reduction inrecognition by HIV-1 positive (polyclonal) sera in ELISA from 100%reactivity to 95%, 91%, and 86% activity when the amino acids leucine,glycine, and leucine, respectively, were specifically removed in astepwise fashion.

Removal of the next two amino acid residues, tryptophan and glycine,which occupy analogous positions within both comparable epitopes forHIV-2 and SIV fitting the sequence formulatryptophan-glycine-cysteine-x-x-x-x-x-cysteine, produced eight andseven-residue oligopeptides (8-mer and 7-mer) that were reactive withonly 64% and 48% of (polyclonal) sera from HIV-positive individuals,respectively. Thus, the essential epitope for immune recognition isdetermined as the 7-amino-acid sequence containing 2 cysteine residues(amino acids 603-609).

The smallest peptide retaining a degree of reactivity (with less than a30% decrease in reactivity when compared to the original 12 amino acidsequence (12-mer)) was that structure with the common amino acidsequence tryptophan-glycine-cysteine-x-x-x-x-x-cysteine, or a nine aminoacid peptide (9-mer). (Gnann, et al., J. Virol. (1987) 61:2639-2641).

Importantly, this region from HIV-1 is immunogenic as well as antigenic.Immunization of New Zealand white rabbits with the synthetic peptideleucine-glycine-leucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine(SEQ NO:28) (amino acid residues 598-609) induced high-titers of bindingpolyclonal antibodies. (Gnann, et al., J. Infect. Dis. (1987)156:261-267.) No data characterizing biological function of the elicitedantibodies were disclosed.

The unique and important characteristic of the described invention isthat: a human B-lymphocyte cell line from an individual exposed to HIV-1(naturally immunized by infection) has been established andimmortalized, and is producing a biologically active fusion-blockinghuman monoclonal antibody directed against an epitope, within thepeptide region described, that being specifically within the amino acidsequenceleucine-glycine-isoleucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine(SEQ NO:2). This is believed to be the first report of human monoclonalantibodies that have been demonstrated to have fusion-blockingbiological activity, as determined by syncytia inhibition assays,against HIV-1 gp41. Although other investigators have developed humanmonoclonal antibodies against gp41, these antibodies have nofusion-blocking biologic activity. (Banapour, et al., J. Immunology(1987) 139:4027-4033; Gorny, et al., Pro. Natl. Acad. Sci. (U.S.A.)(1989) 86:1624-1628; Kawamura, et al., V. International Conference onAIDS (1989), Abstract No. Th.C.O.4, p. 533; Boyer, et al., V.International Conference on AIDS (1989), Abstract No. T.C.P.59, p. 576;Zolla-Pazner, et al., V. Int'l Conf. on AIDS (1989), Abstract No.Th.C.O.10, p. 534).

Furthermore, computer modeling of the HIV-1 envelope protein sequencehas predicted that the two amino acids (Ser-Gly) between the twocysteine residues in question participate in a β turn in the loop,hypothesized to be external to the viral membrane and formed bydisulfide bonding between the two cysteines. (Johnson, et al, supra, p.164, reporting on computer-modeling performed by Modrow, et al., J.Virol. (1987) 61:570-578). Thus, this region, which is also conservedamong numerous isolates of HIV-1 (WMJ1, WMJ2, WMJ3, BH10, ARV2, LAV1A,HAT3), has distinctive biochemical and immunogenic structural propertiesthat allow for a stable antigenic configuration, and, most importantly,also provides the essential biological fusion-associated functionnecessary for the virus to maintain infectiousness.

Specific Recommendation With Regard to Synthetic Peptide Selection As AnImmunogen or Subunit Vaccine Trials

The close resemblance of SIV (STLV-III) with HIV indicates that thefurther development of the experimental models first in rhesus macaquesand African green monkeys will add a great deal to our knowledge inhumans. The analogous 12 amino acid residue peptide (epitope) within thegp32 (SIV 617-628: LNAWGCAFRQVC) (SEQ NO:3) corresponds to both of the12 amino acid residue peptides (or truncated peptides) within the gp41immunodominant epitopes (HIV-1 593-604: LGIWGCSGKLIC (SEQ NO:2), apeptide whose biological function is essential for the maintenance ofviral infectiousness (in the Gnann numbering system this peptidesequence is 598-609) and HIV-2 592-603: LNSWGCAFROVC) (SEQ NO:4) andcould be utilized initially as a subunit vaccine component in an activeimmunization system in non-human primates. Model vaccine strategiescould then be developed for application to a vaccine for human AIDS.

EXAMPLE 1

Production of Cell Lines Synthesizing Human Monoconal Antibodies to HIV

Peripheral blood, collected from a seropositive individual who had hightiters of antibodies to HIV-1, was heparinized, centrifuged to removeplasma, then diluted 1:1 with phosphate buffered saline (PBS), pH 7.4,and subjected to Ficoll-Hypaque gradient centrifugation to obtainmononuclear cells. Approximately 1×10⁷ mononuclear cells at the gradientinterface were recovered, washed thrice with PBS and then incubated for1.5 hours at 37° C. in 3 ml of filtered (0.45 micron) culturesupernatant from EBV transformed marmoset cell line B95-8 containing100,000 transforming units per ml. Cyclosporin A was then added at afinal concentration of 2 micrograms/ml to inhibit suppressor T cells.

Lymphocytes were cultured in 1.5 ml of Iscove Modified Dulbecco's Medium(Iscove, N. N. and Melchers, F., J. Exp. Med. (1978) 147:923-928)supplemented with 10% (vol/vol) fetal bovine serum (HyClone),L-glutamine (2 mM), penicillin (100 units/ml), streptomycin (100micrograms/ml), and fungizone (0.25 micrograms/ml) media for 3 weeks in24 well plates (Costar) at a concentration of 1×10⁶ cells/ml, in anatmosphere of 5% carbon dioxide at 37° C.

After screening for anti-HIV antibody production by a commercialEnzyme-linked Immunosorbent Assay (ELISA) (Genetic Systems, Seattle,Wash.), positive culture wells were cloned by limiting dilution in96-well, U-bottom plates at 1 cell per well on a feeder layer ofirradiated (3000R) human lymphocytes (20,000 cells/well).

An aliquot of lymphoblastoid cells testing positive for anti-HIVproduction was expanded in 25 cm² flasks, 7 ml/flask, (Costar) andcultured for an additional week to a concentration of approximately1×10⁶ cells/ml. Lymphoblastoid cells were then fused by polyethyleneglycol (PEG 50%, MW 1000) with heteromyelomas (HAT sensitive, resistant)in a ratio of 2:1 for preparation of hybridomas. (Bartal, A. H. andHirshaut, Y. Methods of Hybridoma Formation (1987)).

Cell lines with supernatants testing positive for anti-HIV antibodieswere then subcloned twice by limiting dilution at 0.3 cells per well in96-well, U-bottom microtiter plates, to assure monoclonality of theproduced antibody. Each cell line has been cryopreserved in liquidnitrogen to form a master cell bank (MCB). The cells were frozen at aconcentration of 5×10⁶ cells/ml in a freezing solution of Iscove mediasupplemented with 20% fetal bovine serum and 10% dimethylsulfoxide, 1ml/ampule.

EXAMPLE 2

Human Monoclonal Antibody Screening By Enzyme-Linked ImmunosorbentAssays (ELISA) Supernatants from culture wells demonstrating cell growthwere assayed for the presence of human anti-HIV antibodies usingcommercially prepared ELISA plates coated with whole-viral(LAV=lymphadenopathy virus) lysates (e.g., those produced by GeneticSystems, Seattle, Wash.) and envelope glycoprotein, specifically gp160(e.g., those produced by MicroGeneSys, West Haven, Conn.) as targetantigens. The human monoclonal antibody (produced by Clone 3, ATCC No.ATCC CRL 10198) reacts with the envelope (env) gene encoded proteingp160 in ELISA testing, and more specifically gp41, the transmembraneglycoprotein envelope component and is designated Anti-gp41. Thespecificity was determined using the recombinant peptide 121 (p121) asthe target antigen in ELISA testing. The p121 is a polypeptide thatcontains about one-half of the gp41 sequence (the amino terminal half),a major portion of the immunodominant epitopes. (Chang, et al.,Bio/Technology (1985) 3:905-999). The human monoclonal antibody does notreacts with gp120 when tested in a parallel ELISA method against the(external) surface envelope glycoprotein. The specificity results areset forth in Table 1 below. FIG. 4 depicts a linear comparison of thepeptide sequences tested.

                  TABLE ONE                                                       ______________________________________                                        Specificity of Human Monoclonal Antibody                                      ANTI-gp41                                                                     Determined by ELISA                                                                       gp160   gp120   p121  12-mer*                                                                              no Ag                                ______________________________________                                        Human Monoclonal                                                                          +       -       +     +      -                                    Antibody                                                                      Donor Patient                                                                             +       NT      NT    +      -                                    Serum                                                                         Normal Human                                                                              -       NT      NT    -      -                                    Serum                                                                         ______________________________________                                         gp160 = envelope glycoprotein precursor . . . (856 amino acids #001-856)      gp120 = surface envelope glycoprotein . . . (518 amino acids #001-518)        gp41 = transmembrane envelope glyprotein . . . (338 amino acids #519-856)     p121 = recombinant peptide, within gp41 sequence . . . (082 amino acids       #565-646)                                                                     12-mer = conserved 12 amino acid peptide, within gp41 sequence . . . (012     amino acids #598-609)                                                         no Ag = no antigen                                                            + = positive reaction; mean optical density (O.D.) of test greater than       mean O.D. of negative control plus twice the standard deviation. (Barnett     1979, Clin. Lab. Stat., p 124, Little)                                        - = negative reaction; mean optical density (O.D.) of test less than mean     O.D. of negative control plus twice the standard deviation.                   *leucine-glycine-isoleucine-tryptophan-glycine-cysteine-serine-glycine-ly    ine-leucine-isoleucine-cysteine (Seq. No. 2)                                   NT = not tested                                                          

EXAMPLE 3

lmmunoblotting

Additional analyses of specificity were carried out by Western blot (WB)with Biotech Research Labs (Rockville, Md.) and Immunetics (Cambridge,Mass.) HIV-antigen preblotted nitrocellulose membranes using standardtechniques. The human monoclonal antibody reacts with an HIV-1 majorantigen component of 160 kilodaltons (kDa) in viral lysates. It has beendetermined that noncovalently associated tetramers of gp41 represent thenative form of the transmembrane glycoprotein in virions, and thatmonoclonal antibodies preferentially recognize the oligomeric complexesover monomeric gp41 in Western blots (Pinter, et al., J. Virol. (1989)63:2674-2679). FIG. 1 depicts the Western blot testing of the donorpatient plasma and the supernatant from the transformed human B cellline using Biotech Research Labs HIV-antigen preblotted nitrocellulosemembranes. The results demonstrate a positive reaction for thesupernatant corresponding to a band at 160 kDa and co-migrating withgp160.

EXAMPLE 4

Immunoblotting Under Reducing Conditions

FIG. 8 represents the Western blot (WB) testing of the donor patientplasma and the affinity-chromatography purified IgG human monoclonalAnti-gp41 antibody (Clone 3 Antibody) using Immunetics (Cambridge,Mass.) HIV-antigen preblotted nitrocellulose membranes.

In order to reduce disulfide bridges (both intermolecular andintramolecular) that might have formed due to the two invariant cysteineresidues, contained within the gp41, mediating the formation ofoligomers or cyclic antigenic conformations (reference: Berman, P. W.,et al., Journal of Viroloy, August 1989 63:(8), pp. 3489-3498,Expression and Immunogenicity of the Extracellular Domain of the HumanImmunodeficiency Virus Type 1 Envelope Glycoprotein, gp160) thecommercially obtained (Immunetics) HIV-antigen preblotted nitrocellulosestrips were prepared, immediately prior to utilization, with thefollowing modification of the standard procedure described below:

The HIV-antigen preblotted nitrocellulose strips were incubated in PBSbuffer, pH 7.4, with β-mercaptoethanol (1% volume/volume), a reducingagent, at either 37° C. for two hours or 95° C. for two minutes.

Standard immunoblotting technique was then carried out for testing ofthe samples described above, utilizing both reduced and non-reducedpreblotted nitrocellulose strips, in a parallel procedure.

The significant results demonstrated a (weak) positive reaction for thehuman monoclonal antibody (Clone 3 Antibody) corresponding to a band at41 kDa and co-migrating with gp41 only when the HIV-antigen preblottednitrocellulose strip has been reacted with the reducing agentβ-mercaptoethanol. No corresponding reaction band was noted between thehuman monoclonal antibody (Clone 3 Antibody) and the HIV-antigenmigrating at 41 kDa when the HIV antigen preblotted nitrocellulose stripwas not subjected to pretesting reduction treatment withβ-mercaptoethanol.

These data indicate that the human monoclonal antibody (Clone 3Antibody) reacted preferentially with the reduced (linear) gp41fusion-associated octapeptide epitope, GCSGKLIC (SEQ NO:1), inimmunoblotting.

EXAMPLE 5

Affinity Chromatography Isolation and Purification of Human MonoclonalIgG

Aliquots (500 ml volumes) of hybridoma cell culture supernatants (spentcell culture media) were subjected to batch immunoadsorption utilizingSepharose® 4-Fast Flow coupled protein G (Pharmacia, Piscataway, N.J.),a recombinant streptococcal IgG Fc receptor, that has the capacity ofadsorbing 17 mg of human IgG (all subclasses) per ml of gel. Theimmunoadsorbed monoclonal antibody was eluted from the matrix via low pHbuffer (0.1M glycine-HCl, pH 2.4) with rapid neutralization of eluate bycollection into 1.0M TRIS-HCl buffer (pH 9.0) for a final pH of 7.8.

EXAMPLE 6

Determination of Immunoglobulin Class and Subclass

The class and light-chain type of Anti-gp41 monoclonal antibody weredetermined by ELISA. For these assays, commercially-prepared microtiterplates coated with gp160 (MicroGeneSys) were incubated with culturesupernatants (or purified human monoclonal anti-gp41 IgG). The type(class) of antibody was determined with the following horseradishperoxidase-coupled antibodies: goat anti-human IgG (γ chain specific)and goat anti-human IgM (μ chain specific) (Zymed Laboratories, SanFrancisco, Calif.). The subclass of the human monoclonal antibody wasalso analyzed by ELISA with horseradish peroxidase conjugated mousemonoclonal antibodies against the four subclasses (IgG1, IgG2, IgG3,IgG4) of human IgG (Zymed Laboratories, San Francisco, Calif.). Thelight-chain type of Anti-gp41 was determined by exclusion usingperoxidase-labeled mouse monoclonal antibody anti-human κ chain. Theimmunological characterization data of the human monoclonal antibody(Anti-gp41) indicated that the immunoglobulin was of the IgG class,specifically subclass 1, with non-κ, therefore probably λ, light-chaindeterminants.

In an additional immunologic study, in a dot-immunobinding assay (DIBA)on nitrocellulose membranes, as described by Jol-Van der Zijde, et al.,Journal of Immunological Methods (1988) 108:195-203, the humanmonoclonal antibody (Clone 3 Antibody) was demonstrated to react withantiserum (Zymed Laboratories, San Francisco, Calif.) monospecificallydirected against the λ light-chain.

EXAMPLE 7

Ouantitation of Anti-gp41 Human Monoclonal IgG1

IgG quantitation was performed on affinity purified protein by the Lowrytechnique using purified protein (albumin) to produce the standardcurves used in calculations. (Lowry, et al., J. Biol. Chem. (1951)193:265). The concentration of IgG produced by the cell lines varied,but generally reached a maximum at day 9 of culture, ranging from 2-10micrograms/mi.

EXAMPLE 8

Epitope Mapping of Human IgG1 Monoclonal Anti-gp 41 Antibody Binding toTransmembrane Glycoprotein gp41

The wells of Immulon II microELISA plates (Dynatech Industries, McLean,Va.) were coated for a minimum of 12 hours at 4° C. with a solution ofthe synthetic peptide 2,leucine-glycine-isoleucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine(SEQ NO:2) (Cambridge Research Biochemicals, Valley Stream, N.Y.). Thepeptide was solubilized initially with 10% acetic acid and then broughtto a final concentration of 10 μg/ml, pH 6.4 in PBS. One hundredmicroliters of this solution was added per well. In order to minimizepolymerization, β-mercaptoethanol (0.5% vol/vol) was added to thesolution. The wells were emptied of peptide solution, washed thrice with300 microliters PBS containing 0.05% polyoxyethylene (20) sorbitanmonolaurate (e.g., Tween 20, produced by ICI Specialty Chemicals) thentwice with 300 microliters of PBS alone. Excess binding sites wereblocked (quenched) with 200 microliters of PBS containing 1% bovineserum albumin (BSA) per well. Wells containing no peptide were subjectedto the blocking process to be utilized as one parameter of negativecontrols. The wells were incubated with the blocking solution for 3hours at room temperature, then emptied by aspiration, and washed thricewith 300 microliters of PBS. The wells were emptied, dried and thepeptide-coated plates were then stored with desiccant in a sealed bag at4° C. The peptide-coated plates are stable for at least 3 months whenstored in this manner.

To test the binding characteristics of the human monoclonal Anti-gp41antibody with the synthetic peptide, affinity-chromatography purifiedIgG (700 micrograms/ml) was serially diluted in PBS with 0.05%polyoxyethylene (20) sorbitan monolaurate and 1% BSA and reacted (100microliters) in the peptide-coated wells and in wells without antigen(negative control) for 2 hours at 37° C. Normal human serum (negativecontrol) and the donor patient serum were diluted 1:401 and reacted inthe ELISA test against the peptide-coated wells and in wells withoutantigen (negative control). After the initial incubation period, thetest samples and controls were aspirated from the wells which were thenwashed 9 times with 300 microliters of 0.05% polyoxyethylene (20)sorbitan monolaurate in PBS. A solution of 100 microliters of goatanti-human IgG conjugated to horseradish peroxidase was diluted 1:100and added to the wells. After 2 hours at 37° C., the wells were washedagain with 0.05% polyoxyethylene (20) sorbitan monolaurate in PBS, 9times, and then incubated with 100 microliters of tetramethyl-benzidinechromogen in dimethylsulfoxide (DMSO) with buffered hydrogen peroxidesubstrate for 30 minutes at room temperature. The reaction was stoppedby the addition of 100 microliters of 1N sulfuric acid (H₂ SO₄) and theoptical density or absorbance of the solution determined at 490 nm, acolorimetric determination in the visible spectrum.

The results indicate that the human monoclonal antibody bindsspecifically to the gp41 peptide with the amino acid sequenceleucine-glycine-isoleucine-tryptophan-glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine(SEQ NO:2), as did the patient serum. Normal human serum controls werenegative. Additionally, the human monoclonal antibody, patient serum,and normal human serum did not bind to uncoated (no antigen) wells ofthe Immulon II ELISA plate. (See Table 1).

EXAMPLE 9

Epitome Mapping of Human IgG1 Monoclonal Anti-gp41 Antibody Binding toTransmembrane Glycoprotein gp41

The biochemically and immunologically defined human monoclonal antibodyis produced by the Clone 3 cell line (ATCC CRL 10198) and is directedagainst the HIV-1 transmembrane glycoprotein gp41, specifically the 12amino acid peptide (12-mer=Peptide 2),Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-leu-Ile-Cys (SEQ NO:2) (Example 8,Table 1), which has two potential antibody binding regions (epitopes).

Others (Mathiesen, et al., Immunology (1989) 67:1-7) have recentlyinvestigated the binding capacity only of polyolonal human IgG antibodywith two overlapping HIV-1 gp41 peptides (E34/E32; amino acid positions587-608 and 600-618) in order to define the amino acids involved inepitopes and antibodies interactions.

The IgG paratopes showed reactivity to two regions (Mathiesen, id.: page3, first column; page 4, Table 3) within the Peptide 2 sequence,Leu-Gly-Ile-Trp-Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys, (SEQ NO:29) underconsideration in this patent. The two paratopes reacted with the regionswithin the Peptide 2 sequence consisting of the amino acid sequencesIle-Trp-Gly and Gly-Cys-Ser-Gly-Lys-Leu-Ile-Cys (SEQ NO:1).

As shown below in Example 9, Table Two, FIG. 5; Example 10, Table Three,FIG. 6 and FIG. 7, the human monoclonal antibody (Clone 3 antibody)binds preferentially to at least a portion of the amino acid sequenceGly-Cys-Ser-Gly-Lys-Leu-Ile-Cys (SEQ NO:1), an octapeptide (8-mer) whichcontains only one complete epitope of the two antibody binding regionswithin the 12-mer.

The interaction of the fusion blocking human monoclonal antibody withthe identified structural octapeptide sequence (8-mer) (Example 9, TableTwo, FIG. 5) thereby ascribes for the first time an associatedneutralizable physiological function to the immunogenic octapeptideepitope, that being fusion-associated function.

The biological function of the delineated single epitope, consisting ofthe octapeptide amino acid sequence Gly-Cys-Ser-ely-Lys-Leu-Ile-Cys (SEQNO:1) (8-met), denoted as fusion-associated epitope, is not known tohave been described previously in any published or presented scientificpaper. These data are presented in Examples 11 and 18 herein.

The wells of Immulon II microELISA plates (Dynatech Industries, McLean,Va.) were coated for a minimum of 12 hours at 4° C. with a solution ofthe synthetic peptide 6120 (linear),glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine-threonine-threonine-alanine-valine-proline-tryptophan-asparagine-alanine-serine(SEQ NO:5). The cyclic peptide 6120 (IAF BioChem International, Inc.,Montreal, Canada) was solubilized initially with 10% acetic acid andthen brought to a final concentration of 10 micrograms/ml, pH 6.4 inPBS. In order to minimize polymerization and cyclization and to reducethe cyclic peptide, β-mercaptoethanol (1% vol/vol) was incubated withthe solution for 1 hour at 37° C. Then, one hundred microliters of thisreduced, linear peptide solution was added per well. The wells wereemptied of peptide solution, washed thrice with 300 microliters PBScontaining 0.05% polyoxyethylene (20) sorbitan monolaurate (e.g., Tween20, produced by ICI Specialty Chemicals) then twice with 300 microlitersof PBS alone. Excess binding sites were blocked (quenched) with 200microliters of PBS containing 1% bovine serum albumin (BSA) per well.Wells containing no peptide were subjected to the blocking process to beutilized as one parameter of negative controls. The wells were incubatedwith the blocking solution for 3 hours at room temperature, then emptiedby aspiration, and washed thrice with 300 microliters of PBS. The wellswere emptied, dried and the peptide-coated plates were then stored withdesiccant in a sealed bag at 4° C. The peptide-coated plates were stablefor at least 3 months when stored in this manner.

To test the binding characteristics of the human monoclonal Anti-gp41antibody with the synthetic peptide 6120, affinity-chromatographypurified IgG, (1 mg/ml) was serially diluted in PBS with 0.05%polyoxyethylene (20) sorbitan monolaurate and 1% BSA and reacted (100microliters) in the peptide-coated wells and in wells without antigen(negative control) for 2 hours at 37° C. Normal human serum (negativecontrol) and the donor patient serum were diluted 1:401 and reacted inthe ELISA test against the peptide-coated wells and in wells withoutantigen (negative control). After the initial incubation period, thetest samples and controls were aspirated from the wells which were thenwashed 9 times with 300 microliters of 0.05% polyoxyethylene (20)sorbitan monolaurate in PBS. A Solution of 100 microliters of goatanti-human IgG conjugated to horseradish peroxidase was diluted 1:100and added to the wells. After 2 hours at 37° C., the wells were washedagain with 0.05% polyoxyethylene (20) sorbitan monolaurate in PBS, 9times, and then incubated with 100 microliters of tetramethyl-benzidinechromogen in dimethylsulfoxide (DMSO) with buffered hydrogen peroxidesubstrate for 30 minutes at room temperature. The reaction was stoppedby the addition of 100 microliters of 1N sulfuric acid (H₂ SO₄) and theoptical density or absorbance of the solution determined at 490 nm, acolorimetric determination in the visible spectrum.

The results indicate that the human monoclonal antibody (Clone 3Antibody) binds specifically to the gp41 peptide with the amino acidsequenceglycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine-threonine-threonine-alanine-valine-proline-tryptophan-asparagine-alanine-serine(SEQ NO:5) (reduced, linear, non cyclic), as did the donor patientserum. Normal human serum controls were negative. (Table Two).

The amino acid sequence common to both Peptide 2 (12-mer) and Peptide6120 and to which Clone 3 human monoclonal antibody binds is theoctapeptide (8-mer)glycine-cysteine-serine-glycine-lysine-leucine-isoleucine-cysteine (SEQNO:1). (See FIG. 5).

                  TABLE TWO                                                       ______________________________________                                        Specificity of Human Monoclonal Antibody                                      (Clone 3 Antibody)                                                            ANTI-gp41                                                                     Determined by ELISA                                                                             Peptide 2*                                                                              Peptide                                                             (12-mer)  6120     no Ag                                    ______________________________________                                        Human Monoclonal Antibody                                                                       +         +        -                                        Donor Patient Serum                                                                             +         +        -                                        Normal Human Serum                                                                              -         -        -                                        ______________________________________                                         *Peptide 2(12mer) = 12 amino acid peptide within gp41 sequence . . .          (amino acids #598-609)                                                        Peptide 6120 = 17 amino acid peptide with gp41 sequence . . . (amino acid     #602-618)                                                                     no Ag = no antigen                                                            + = positive reaction; mean optical density (O.D.) of test greater than       mean O.D. of negative control plus twice the standard deviation. (Barnett     1979, Clin. Lab. Stat., p. 124, Little)                                       - = negative reaction; mean optical density (O.D.) of test less than mean     O.D. of negative control plus twice the standard deviation.              

EXAMPLE 10

Fine Epitope Mapping of Human IgG1 Monoclonal Anti-gp41 Antibody byELISA Using Synthetic Pentadecapeptides (15 Amino Acid Peptides)

Pentadecapeptides, sequentially overlapping by 10 amino acids weresynthesized on the basis of HTLV-IIIB, clone B10 (Ratner et al., Nature(1985) 313:277), for gp41 peptides 232-240 (amino acids 572-626). Thesewere synthesized according to the solid-phase method of Merrifield, J.Amer. Chem. Soc. (1963) 85:2149, modified by Houghten, Proc. Natl. Acad.Sci. U.S.A. (1985) 82:5131. For human monoclonal antibody ELISAs,microwell plates (Nunc Immunoplate I) were coated with 1 microgram ofpeptide/microwell. Sera samples and affinity column (Protein G) purifiedhuman monoclonal antibody were assayed in dilutions of 1:50 for 105minutes at 37° C. Mouse antibodies to IgG were used to bind to human IgGand subsequently detected by HRPO-conjugated anti-mouse Ig. A colorreaction was obtained with ortho-phenylenediamine (OPD) and the opticaldensity (OD) at 490 nm was recorded. Blanks and HIV seronegativecontrols were included in all plates. Sera and human monoclonal antibodygiving greater than 3 times the mean OD of negative controls (alwaysmore than mean+3SD) were scored as positive for IgG and peptide.

The results indicate that the human monoclonal antibody (Clone 3Antibody) binds specifically to Peptide 237 and Peptide 238, as did thedonor patient serum. Normal human serum controls were negative. (TableThree and FIG. 6). The decapeptide sequence common to both Peptide 237and Peptide 238 contains the octapeptide amino acid sequence, thefusion-associated epitope (FIG. 7).

                                      TABLE THREE                                 __________________________________________________________________________    Specificity of Human Monoclonal Antibody                                      (Clone 3 Antibody)                                                            ANTI-gp41                                                                     Determined by ELISA                                                                           Peptide 235                                                                         Peptide 236                                                                             Peptide 237                                                                         Peptide 238                                                                             Peptide                                                                             Peptide                 __________________________________________________________________________                                                          240                     Human Monoclonal Antibody                                                                     0.01* 0.01      0.33  0.56      0.12  0                       Donor Patient Serum                                                                           0.25  1.00      1.70  1.75      0.70  0.25                    Normal Human Serum                                                                            0     0         0     0         0     0                       __________________________________________________________________________    Sera samples diluted 1:50; human monoclonal antibody concentration = 20       μg/ml)                                                                     Peptide 235 = (15 amino acids #587-601)                                                            AVERYLKDQQLLGIW (Seq. No. 6)                             Peptide 236 = (15 amino acids #592-606)                                                            LKDQQLLGIWGCSGK (Seq. No. 7)                             Peptide 237 = (15 amino acids #597-611)                                                            LLGIWGCSGKLICTT (Seq. No. 8)                             Peptide 238 = (15 amino acids #602-616)                                                            GCSGKLICTTAVPWN (Seq. No. 9)                             Peptide 239 = (15 amino acids #607-621)                                                            LICTTAVPWNASWSN (Seq. No. 10)                            Peptide 240 = (15 amino acids #612-626)                                                            AVPWNASWSNKSLEQ (Seq. No. 11)                            *Absorbance = Optical Density (OD) at 490 nm                                  The decapeptide amino acid sequence common to both Peptide 237 and            Peptide 238, the peptides for which                                           the Human Monoclonal Antibody (Clone 3 Antibody) had the greatest             binding, is underscored.                                                      The fusion-associated epitope, GCSGKLIC, is an octapeptide contained          within the decapeptide.                                                   

The biological reactivity of the human monoclonal antibody (Clone 3Antibody) has been demonstrated in inhibition of syncytia formationassays wherein the neutralization capacity of the human monoclonalantibody is characterized by the blocking of fusion between HIV-1infected and uninfected human cells (Example 11, Table Four, FIG. 2) andby neutralization of free HIV-1 (SF2) infectivity as presented below inExample 18 and Table Five.

EXAMPLE 11

Quantitative Synctium-Forming Microassay for the Detection of HumanImmunodeficiency Virus Neutralizing Antibody

Traditionally, retroviruses, including HIV-1, can be assayed directly bya number of simple focus-forming or syncytium-forming assays. (Nara, etal., AIDS Research and Human Retroviruses (1987) 3:283-302; Putney, etal., Science (1986) 234:1392-1395). These assays are very sensitive,simple, relatively rapid, and allow for ready biological assessmentconcerning infectivity of virus under various in vitro conditions.Additionally, various virus-envelope associated properties such asinterference by inhibitory agents or neutralization by antibodies can bestudied.

The human monoclonal antibody anti-gp41 was tested in thesyncytium-forming microassay procedure, as described by Putney, toascertain HIV-1 neutralization capabilities as demonstrated by fusioninhibition. The microtiter syncytium-forming assay utilized a clone ofCEM (CD4+) cells chronically infected with the HIV-1 isolate HTLV-IIIB,and MOLT-4 (CD4+) cells. CEM cell stocks stably infected with HTLV-IIIB,yet not susceptible to the cytopathological effects of the virus, wereused as the infected partner and MOLT-4 cells were used as theuninfected partner. Cells were washed once in growth media and cellconcentrations were adjusted to 0.125×10⁶ cells/ml and 1.75×10⁶ cells/mlfor CEM and MOLT-4 cells, respectively. Ninety-six well, half-areaplates were used in the assay. Anti-gp41 antibody solution was added tohalf-area wells in a volume of 10-20 microliters. Forty microliters ofeach cell solution was then added to the well, resulting in a ratio ofapproximately 5,000 HTLV-IIIB-infected CEM cells to 70,000 uninfectedMOLT-4 cells per well. Plates were incubated at 37° C. in an atmosphereof 5% CO₂ for 20-24 hours.

Syncytia formation of cells, or giant cell formation, was enumeratedusing an inverted microscope at 40× magnification. The number of giantcells, defined as multinucleated fused cells being 5 times the diameterof input cells, were scored as the number of syncytium-forming units perwell.

The results of fusion inhibition by Anti-gp41 are quantitativelyreported. The human monoclonal antibody concentration with thecorresponding syncytium-forming units (SFUs) observed are presented inTable Four below, where V_(o) is the total number of virus induced SFUsper well in the absence of antibody and V_(n) is the number of SFUs perwell in the presence of antibody, in doubling dilutions of (decreasingconcentration).

                  TABLE FOUR                                                      ______________________________________                                        Syncytium-Forming Microassay                                                  HUMAN                                                                         Monoclonal Antibody*                                                                           Number of Syncytium-                                         Dilution         Forming Units (SFU) V.sub.n                                  ______________________________________                                         1:5 (140 μg/ml)                                                                             6                                                           1:10 (70 μg/ml)                                                                             22                                                           1:20 (35 μg/ml)                                                                             35                                                           1:40 (17.5 μg/ml)                                                                           39                                                           1:80 (8.75 μg/ml)                                                                           33                                                           Control (no antibody) (V.sub.o)                                                                36                                                           ______________________________________                                         SFU average of replicates of quadruplicate determinations (V.sub.n and        V.sub.o)                                                                      *700 micrograms/ml = (protein concentration neat)                        

The human monoclonal Anti-gp41 antibody at a 1:5 dilution (the minimumdilution allowed and therefore the maximum antibody concentration testedin the microassay system) decreased in number the formation ofsyncytium-forming units between HIV-1 (HTLV-IIIB) infected CD4+ cellsand uninfected target CD4+ (MOLT-4) cells from 36 (V_(o)) to 6 (V_(n))for a fusion inhibition of 83%. Replicate tests were performed inquadruplicates, and fusion inhibition percentage values were calculatedfrom the reduction in virus-induced, syncytial-forming units,represented as (V_(o) -V_(n)), obtained in the presence of two-folddilutions of human monoclonal Anti-gp41, divided by the number of totalvirus induced SFUs added (V_(o)).

In the microassay system, the concentration of the affinity column(Protein G) purified human monoclonal Anti-gp41 IgG1 that resulted in an83% inhibition in syncytium-formation was 140 micrograms/ml, aphysiological concentration. The 50% SFU inhibition point (Vn=18) wasobtained at a human monoclonal Anti-gp41 IgG1 antibody concentration of˜88 micrograms/ml. (See FIG. 2).

EXAMPLE 12

Therapeutic Uses For Anti-gp41

The antibody of the invention may be used therapeutically, as describedbelow, in the modality of passive immunotherapy. Human monoclonalantibodies with the proper biological properties are useful directly astherapeutic agents. Data to support the efficacy and delineation of thetherapeutic protocols for passive immunotherapy in other primates(chimpanzees) have been published, for it has been determined thatneutralization of in vivo HIV-1 infectivity can be mediated by in vitroneutralizing antibody directed against the hypervariable loop of theviral envelope (gp120). Chimpanzee-derived polyclonal antibodies wereutilized in the protocol. (Emini, et al., V. International Conference onAIDS (1989), Abstract No. Th.C.O.30, p. 538). By administering anappropriate human monoclonal antibody to patients who lack neutralizingantibodies against the envelope epitope within gp41, passiveimmunotherapy can be provided.

In a parallel human study, data from recent clinical trials (Jackson, etal., Lancet (1988) 2:647-652; Karpas, A., Proc. Natl. Acad. of Sciences(U.S.A.) (1988) 85:9234-9237) have demonstrated that passiveimmunization improved the status of patients with advanced (symptomatic)AIDS. In those trials passive immunization was accomplished bytransfusing plasmas containing antibodies from asymptomatic AIDSpatients into the symptomatic AIDS recipients.

For example, the passive immunization method against hepatitis B virushas been utilized in humans, when clinically indicated, as routineeffective measures of post-viral exposure prophylaxis, wherein hepatitisB immune globulin is administered to the at-risk recipient at a dose of0.06 ml/kg IM (Center for Disease Control, Department of Health andHuman Services. "Recommendations for Protection Against Viral Hepatitis.Recommendation of the Immunization Practices Advisory Committee." MMWR(1990) 39:1-26).

When a neutralizing agent, such as a human monoclonal antibody, is usedin passive immunotherapy, the protocol regimen can parallel that for theadministration of hepatitis B immune globulin (e.g., H-BIG®, a solutionof human immunoglobulin obtained from pooled venous plasma ofindividuals with high titers of antibody to the hepatitis B surfaceantigen, prepared by Abbott Laboratories, North Chicago, Ill. 60064).

Neutralizing antibodies and antibodies mediating antibody-dependentcellular cytotoxicity (ADCC) to HIV represent important responses sought(for effective passive immunotherapy and) in an effective HIV vaccinefor active immunization. (Weiss, et al., Nature (1985) 316:69-71;Robert-Guroff, et al., supra, 72; Ho, et al., J. Virol. (1987) 61:2024;Cheng-Mayer, et al., Proc. Natl. Acad. Sci. (U.S.A.) (1988) 85:2815; andRook, et al., J. Immunol. (1987) 138: 1064; Ljunggren, et al., (1987)139:2263; Ojo-Amaize, et al., 2458; Blumberg, et al., J. Infect. Dis.(1987) 156:878; Shepp, et al., id. (1988) 157:1260; Tyler, et al., VInternational Conference on AIDS (1989), Abstract No. T.C.O.33, p. 521).

It is significant, therefore, that Clone 3 Antibody, which binds to thegp41 fusion-associated epitope (GCSGKLIC) (SEQ NO:1), can both preventfusion of virus-infected cells and neutralize infectivity of free virusparticles, as demonstrated by biological assays presented in Example 11and Example 18, respectively.

Alternatively, the monoclonal antibody can be bound to a toxin such asdeglycosylated ricin A (dgA) chain to form an immunotoxin (IT). Methodsfor producing immunotoxins of antibodies are well known. The A chain ofricin may be chemically deglycosylated to prevent any immunotoxinsubsequently formed from binding to the parenchymal and nonparenchymalcells of the liver through mannose receptors. To produce the derivatizedantibody which can then be coupled to dgA, N-succinimidyl3-(2-pyridyldithio) propionate dissolved in dimethyl-formamide may beadded to a solution of the antibody (5 mg/ml) in 0.1M sodium phosphatebuffer with 0.003M Na₂ EDTA (disodium ethylenediamine tetracetic acid)(pH 7.5) to a final concentration of 1 mM. After 30 minutes at roomtemperature, the solution may be desalted on a column of Sephadex G-25.The derivatized immunoglobulin may then be added to the dgA chainsolution at a ratio of 1.3 mg of deglycosylated A chain to 1 mg of IgGand maintained for 2 hours at 25° C., followed by overnight at 4° C. Theresultant IT-dgA (immunotoxin dgA chain-antibody) may then be purifiedon Sephacryl ACA-44. (Till, et al., Proc. Natl. Acad. Sci. (U.S.A.)(1989) 86:1987-1991).

Only HIV-1-infected cells express viral proteins on their surface.Consequently, only those cells infected with the virus will expressgp41. Since the monoclonal antibody of this invention binds to gp41, themonoclonal antibody will be able to target the toxin to only those cellsinfected with the virus. Additionally, gp41 is a highly conservedpeptide, thereby making the therapy described practicable. Thisparticular monoclonal antibody is also able to inhibit cellular fusionbetween HIV-1-infected and uninfected cells, thus effecting dualpurposes when conjugated to the deglycosylated ricin A chain.

Also included within the scope of the invention are useful bindingfragments of the described monoclonal antibody, such as Fab, F(ab')₂,and Fv fragments. The antibody fragments are obtained by conventionaltechniques. Useful binding fragments may be prepared by peptidasedigestion of the antibody using papain or pepsin. Consequently, theantibodies, or fragments thereof, may be injected either with or withouta conjugated material for use in treating individuals already infectedwith AIDS.

EXAMPLE 13

Diagnostic Uses for Anti-g41

Another example of a technique in which the monoclonal antibody and thebiological reactive fragments of the invention may be employed is inimmunodiagnostic assays involving antigen-antibody reactions. The assaysmay be homogeneous or heterogeneous. In a homogeneous assay approach,the specimen may be biological fluid such as serum, urine, and the likeor the specimen may be lysed and clarified to remove debris. Theantibody, for example, can be used to measure the concentration of itscognate antigen in the serum of patients. The immunological reactionusually involves the specific antibody, a labeled analyte, and thesample of interest. The signal arising from the label is modified,directly or indirectly, upon the binding of the antibody to the labeledanalyte. Both immunological reaction and detection of the extent thereofare carried out in a homogeneous solution. Immunochemical labels whichmay be employed include free radicals, fluorescent dyes, enzymes,bacteriophages, coenzymes, and so forth.

In a heterogenous assay approach, the reagents are usually the specimen,the specific antibody, and means for producing a detectable signal. Thespecimen is generally placed on a support, such as a plate or slide, andcontacted with the antibody in a liquid phase. The support is thenseparated from the liquid phase and either the support phase or theliquid phase is examined for a detectable signal employing means forproducing such signal. The signal is related to the presence of theanalyte in the specimen. Means for producing a detectable signalincludes the use of radioactive labels, fluorescent compounds, enzymes,and so forth. Exemplary heterogeneous immunoassays are theradioimmunoassay, immunofluorescence methods, enzyme-linkedimmunoassays, and the like.

For a more detailed discussion of the above immunoassay techniques, see"Enzyme-Immunoassay," by Edward T. Maggio, CRC Press, Inc., Boca Raton,Fla., 1980. See also, for example, U.S. Pat. Nos. 3,690,834; 3,791,932;3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074;3,984,533; 3,966,345; and 4,098,876, which listing is not intended to beexhaustive. Methods for conjugating labels to antibodies and antibodyfragments are well known in the art. Such methods may be found in U.S.Pat. Nos. 4,220,450; 4,235,869; 3,935,974; and 3,966,345. Anotherexample of a technique in which the monoclonal antibody of the inventionmay be employed is immunoperoxidase labeling. (Sternberger,Immunocytochemistry (1979) pp. 104-169). Alternatively, the monoclonalantibody may be bound to a radioactive material or to a drug to form aradiopharmaceutical or pharmaceutical, respectively. (Carrasquillo, etal., Cancer Treatment Reports (1984) 68:317-328).

EXAMPLE 14

Therapeutic Uses of Fusion-Associated Epitope of Human ImmunodeficiencyVirus-1 (HIV-1)

With regard to active immunization, technology is now available forsynthesis of vaccines derived from short amino acid sequences on thesurface of a virus or other pathogen. Such small peptide sequences arecapable of representing the immunogenic counterpart in the entirepathogen and of stimulating effective neutralizing antibody production.These synthetic vaccines are potentially "medically ideal" reagents,whose clinical applicability has now been studied for almost a decade.(Lerner, R. A., et al., Hospital Practice (1981) 16:55-62, December1981, Vol. 16, No. 12, pp. 55-62).

The synthetic vaccine approach has been studied in SIV infection inmacaques, on the basis of immunological studies with a set of SIVpeptides chosen for hydrophilicity and conservation epitopes analogousto HIV gp120 and gp41. The immunogenic potential of a mixture of four ofthese epitopes (two from gp120 and two from gp41) was tested inmacaques. One of the two SIV transmembrane synthetic peptide sequences,corresponding to the HIV-1 transmembrane gp41 amino acid sequence (aminoacids 589-609) that contains the neutralizable fusion-associated epitope(amino acids 602-609) considered in this patent as anantigenic/immunogenic peptide of HIV-1, was one of the four subunitsynthetic vaccine components utilized to successfully immunize themacaques against SIV challenge. (Shafferman, A., et al., Cold SpringHarbor Laboratory Symposium (September 1990) p. 90).

Other viral pathogens for which synthetic peptide vaccines have beensuccessfully developed include foot-and-mouth disease of cattle (Bittle,J. L., et al., Nature (1982) 298:30; Vaccine 89, Cold Spring HarborLaboratory Symposium (September 1989) p. 449) and hepatitis B virus inhumans, for which a vaccine consists of a single viral protein, thesurface antigen prepared by recombinant DNA technology. (Schild, G. C.,et al., Lancet (1990) 335:1081).

The active immunization method, utilizing the HIV-1 fusion-associatedepitope (peptide) can parallel that standardized for hepatitis B,wherein, for example, 20 micrograms of recombinant peptide is theinoculum per each of three inoculations (inoculum=1 ml) (IM-deltoidmuscle), at time intervals of initial, one, and six months after thefirst dose (e.g., Engerix-B®, a hepatitis B recombinant peptide vaccine,manufactured by SmithKline Biologicals, Rixensart, Belgium, distributedby Smith Kline & French Laboratories, Division of SmithKline BeckmanCorporation, Philadelphia, Pa. 19101).

Because it is not known whether the predominant route of initial HIV-1infection in humans is by cell-associated virus or cell-free virus, itis important that any vaccine antibody response based on the gp41fusion-associated epitope (GCSGKLIC) (SEQ NO:1) be able to block bothinfectious pathways. Again, it is significant, therefore, that Clone 3Antibody, elicited by the native antigen identified in fine epitopemapping as in the octapeptide represented by the amino acid sequenceGCSGKLIC (SEQ NO:1), can both prevent fusion of virus-infected cells andneutralize infectivity of free virus particles, as demonstrated bybiological assays presented in Example 11 and Example 18, respectively.

Therefore, therapeutic measures capable of boosting the (decreasing)neutralizing antibody titer of individuals already infected with thehuman immunodeficiency virus-1 (HIV-1), eliciting high-titerneutralizing antibodies (i.e., active immunotherapy), or increasing(augmenting) neutralizing antibodies (i.e., passive immunotherapy) inindividuals at risk would prove beneficial in preventing new infectionor in controlling viral spread in vivo, (Robert-Guroff, et al., AIDSResearch and Human Retroviruses (1988) 3:343-350), thereby preventingthe disease progression to frank AIDS.

EXAMPLE 15

Prognostic Uses of Fusion-Associated Epitope of Human ImmunodeficiecyVirus-1 (HIV-1) and Anti-gp41 (Clone 3 Antibody)

The peptide can be used as a prognostic tool to measure theconcentration of the protecting antibody in the patient's serum. Asmentioned previously, there is a correlation between level of protectiveantibody and the advancement of the disease. As protective antibodylevel decreases, the disease-state progresses.

Patient samples such as plasma, cerebral spinal fluid, secretions, orexcretions may be collected for testing. The samples could then betested in an in vitro ELISA for quantitating and detecting antibodyagainst the peptide. Microtiter plates coated with the peptide could beused in the screening. The patient samples could be incubated on thepeptide-coated plates in various dilutions for times sufficient to allowbinding to occur. An anti-human antibody labeled either radioactively orenzymatically for subsequent detection could then be added to the wells.The simultaneous running of a standard curve with known antibody amountswould enable quantitation of the antibody in the patient samples.

Conversely, the human monoclonal antibody can be utilized to detect,monitor, and quantitate the concentration of the respectiveantigen/virus in the biological fluids listed above, or in acell-associated state.

AIDS is caused by the retrovirus Human Immunodeficiency Virus-1 (HIV-1).HIV-1 infection is a chronic disease. The time between infection and thedevelopment of clinical AIDS probably averages 8 to 10 years. Laboratorytests for Human Immunodeficiency Virus-1 infection are commonlyperformed for two reasons.

First, diagnostic tests for the detection of the presence of HIV-1antibodies can determine whether a person has been infected with thevirus. Frequently the virus itself is not detected in patients who areseropositive for HIV-1 antibodies. In early stages of the disease mostindividuals who have been infected with HIV-1 do not develop clinicalsymptoms caused by the associated immunosuppression that graduallyensues and therefore most infected individuals are asymptomatic withregard to demonstrating signs of the illness.

Second, prognostic tests should be conducted to estimate the stage andactivity of HIV-1 disease. Therefore, prognostic tests are important inidentifying patients who might benefit from prophylactic therapy againstHIV-1 disease progression.

A number of tests have been used to help estimate the rate of diseaseprogression and the stage of disease. Monitoring these laboratoryfindings are helpful in patient clinical management. Quantitative testsinclude CD4+ (T-helper) lymphocytes counts, p24 antigen levels, andbeta₂ -microglobulin levels.

A decrease in average antibody titers (concentration) has beenclinically observed in late stages of infection, particularly withregard to antibodies directed against the HIV-1 envelope epitopes andspecifically against the TM gp41 region containing the amino acidsequence against which the herein described human monoclonal antibody(Clone 3 Antibody) is biologically reactive. (Shafferman, et al., AIDSResearch and Human Retroviruses (1989) 5:33-39; Chiodi, et al., J. Med.Virol. (1987) 23:1-9; McPhee, et al., FEBS Lett. (1988) 233:393-396).

Specifically and significantly, it has been determined recently that alow IgG antibody titer (concentration) against a gp41 peptide (JB7)represented by amino acids CSGKLICTT (SEQ NO:12) (603-611,Wain-Hobson/Gnann numbering system) was found to be associated with arapid disease progression to frank AIDS. (Broliden, et al., AIDS (1989September) 3:577-582). The clinical correlation and the significant dataare presented below:

Sera from children that had progressed to frank AIDS (symptomatic)contained almost no antibody reactivity against the indicated region ofgp41, CSGKLICTT (SEQ NO:12). Only one out of seven sera from thechildren with frank AIDS showed antibody reactivity to CSGKLICTT (SEQNO:12), compared with 12 out of 15 of the rest of the group infectedwith HIV-1, yet without symptoms of AIDS (asymptomatic).

This significant difference (P less than 0.025, chi square) regardingthe concentration of antibodies against the gp41 peptide JB7, could notbe seen in peptides representing other parts of gp41, i.e., JB2 and JB4.

The amino acid sequences (single letter abbreviations) are presented forgp41 peptide JB2 and gp41 peptide JB4, for comparison to the amino acidsequence for gp41 peptide JB7, a nine amino acid peptide which contains7 of the 8 amino acids of the fusion-associated epitope. Additionally,the octapeptide amino acid sequence for the gp41 fusion-associatedepitope, which contains 7 of the 9 amino acids of the JB7 peptide, isalso provided below:

    __________________________________________________________________________    JB2   .sup.578 IKQLQARILAVERYLKDQQLLGIWG.sup.602 (Wain-Hobson/Gnann)                (Seq. No. 13)                                                           JB4   .sup.599 GIWGCSGKLICTTAVPWNAS.sup.618 (Seq. No. 14)                     JB7   .sup.603 CSGKLICTT.sup.611 (Seq. No. 12)                                Fusion-Associated Epitope.sup.602 GCSGKLIC.sup.609 (Seq. No.                  __________________________________________________________________________    1)                                                                        

However, anti-JB7 (antibody) response probably has no protectivefunction against infection (i.e., absolute prevention of transmission ofHIV-1) since the frequency of anti-JB7 (antibody) reactivity in theuninfected group (without HIV-1) was also low. The data indicated thatonly four sera from 19 of the uninfected children less than 6 months ofage reacted with JB7, i.e., demonstrated the presence of anti-JB7antibodies.

A similar difference of reactivity would probably be possible foridentifying other significant (neutralizable) epitopes contained withingp41, with the corresponding (neutralizing) antibodies.

Fusion-Associated Epitope--The peptide can be synthesized according tothe solid-phase method of Merrifield (1963) modified by Houghten (1985).For ELISA quantitation of human antibody directed against thefusion-associated epitope, microwell plates (NUNC Immunoplate 1) arecoated at 4° C. overnight (or 3 hours at room temperature) with 1microgram of peptide/microwell. The wells are washed three times withphosphate buffered saline (PBS) and are then incubated with 200microliters of PBS containing 1% bovine serum albumin (BSA) per well atroom temperature for 3 hours to block nonspecific protein binding sites.The wells are then emptied by aspiration, and washed three times with300 microliters PBS containing 0.05% by volume polyoxyethylene (20)sorbitan monolaurate (e.g., Tween 20, produced by ICI SpecialtyChemicals), then twice with 300 microliters of PBS alone. The wells areemptied, dried and the fusion-associated epitope/peptide coated platesare then stored with desiccant in a sealed bag at 4° C.

The test fluid (e.g., blood serum from a human patient or normalindividual) is diluted with PBS containing 20% by volume normal goatserum, 1% by weight BSA and 0.05% by volume Tween 20 at dilutions of1:50 (or 1:500), volume to volume.

In order to quantitate the human antibody in the test sera directedagainst the fusion-associated epitope, 100 microliters of the dilutedsera are added to each well and allowed to react for 2 hours at 37° C.To construct a standard curve in order to calculate the concentrationsin test sera of human antibodies directed against the fusion-associatedepitope, a known concentration of affinity-chromatography purified Clone3 Antibody (IgG1) is serially diluted and concomitantly reacted (100microliters) in the peptide-coated wells. The wells are then washed fivetimes with 0.05% by volume Tween 20 in PBS in order to remove Unboundantibodies. A solution of 100 microliters of horseradish peroxidaseconjugated goat anti-human IgG at a dilution of 1:100 is used as asecond antibody tracer to bind with the human antibody-antigen complexformed in positive wells After 2 hours at 37° C., the wells are washedthree times with 0.05% by volume Tween in PBS, then incubated with 100microliters of tetramethylbenzidine chromogen in dimethylsulfoxide(DMSO) with buffered hydrogen peroxide substrate for 30 minutes at roomtemperature. The reaction is stopped by the addition of 100 microlitersof 1N sulfuric acid (H₂ SO₄) and the optical density or absorbance ofthe solution determined at 490 nm, a colorimetric determination in thevisible spectrum. Assays are performed in duplicate serum samples fromnormal individuals or from patients with diseases unrelated to HIV-1infection used as negative controls. Absorbance readings greater thanthe cutoff value of A₄₉₀ =0.12, (about 3 times the mean A₄₉₀ value ofnormal serum control), are recorded as positive.

A prognostic test kit for quantitation of human antibodies directedagainst the fusion-associated epitope of HIV-1 can be constructed. Thetest kit comprises a compartmented enclosure containing multiple 96-wellplates coated prior to use with 1 microgram per well of thefusion-associated epitope/peptide of the present invention. The kitfurther comprises materials for enzyme detection in separate sealedcontainers consisting of: (1) normal human serum, as negative control;(2) quantitated affinity-chromatography purified Clone 3 Antibody (humanmonoclonal anti-fusion-associated epitope antibody directed againstHIV-1 gp41), as positive control and for construction of a standardcurve; (3) normal goat serum; (4) peroxidase labeled-goat antihuman IgG;(5) color change indicator consisting of tetramethylbenzidine chromogenin dimethylsulfoxide (DMSO) with buffered hydrogen peroxide substrate;and (6) 1N sulfuric acid (H₂ SO₄). The procedure-described above is tobe followed.

EXAMPLE 16

Development and Use of Synthetic Inhibitory Peptide

Since the amino acid sequence of the fusion-associated epitope is known,it is possible to synthetically develop a complementary peptide capableof binding to the epitope and thereby capable of blocking fusion. Thedevelopment of a "synthetic inhibitory peptide" is facilitated in thisinstance since the tertiary structure of the epitope has been predictedthrough computer analysis. (Modrow, et al., J. virol., (1987)61:570-578; Navia, et al., V International Conference on AIDS (1989)Abstract No. M.C.O.23, p. 513; Debouck, et al., V InternationalConference on AIDS (1989), Abstract No. T.C.O.11, p. 517).

The amino acid sequence and tertiary structure of the epitope may beinput as data into a computer program with 3-dimensional modelingcapabilities. Several models of complementary peptides may then begenerated. Peptide sequences consisting of the complementary peptidesmay then be synthesized and tested for fusion inhibition capacity, suchas in a syncytium-forming assay. Those peptides found to inhibit fusionmay then be produced on a larger scale for therapeutic purposes. Suchpeptides may be administered orally, intramuscularly, or intravenously.

Specifically, an example of the complementary synthetic inhibitorypeptide on gp120 (amino acids 107-134, Myers) that has the biochemicalproperty to form amphipathic helices with three charged amino acidresidue contacts (shown below and underscored) and complementaryhydrophobic residues on the gp41 (amino acids 584-611,Wain-Hobson/Gnann) and consequently inhibit the function of thefusion-associated epitope on gp41 (amino acid residues 602-609,Wain-Hobson/Gnann), would be, as deduced from the data presented byMcPhee, et al., Cold Spring Harbor Symposium (1988), p. 17, and Modrow,S., id.:

    107.sub.D-I-I-S-L-W-D-Q-S-L-K-P-C-V-K-L-T-P-L-C-V-S-L-K-C-T-D-L 134(Myers)(SEQ NO:15)

(single letter abbreviations representing amino acid residues 107-134,Myers) .

The predicted three charged amino acid residue contacts (shown below andunderscored) and the complementary hydrophobic amino acid residues thatform part of the putative contact region between gp120 (amino acids107-134, Myers) and gp41 (amino acids 584-611, Wain-Hobson/Gnann) andthe relation to the fusion-associated epitope (shown below in bold type)on gp41 (amino acids 602-609, Wain-Hobson/Gnann), are represented in thecomparison below.

    .sub.gp120 105.sub.H-E-D-I-I-S-L-W-D-Q-S-L-K-P-C-V-K-L-T-P-L-C-V-S-L-K-C-T-D-L 134(Myers)                                                (SEQ NO:16)

    .sub.gp41 582.sub.Q-A-R-I-L-A-V-E-R-Y-L-K-D-Q-Q-L-L-G-I-W-G-C-S-G-K-L-I-C-T-T 611(Gnann)                                                (SEQ NO:17)

Thus, with regard to the synthetic inhibitory peptide (neutralizingagent), the complementary synthetic inhibitory peptide sequence may bedetermined as presented above.

Additional data indicating that the antiviral synthetic inhibitorypeptides against HIV-1 can be synthesized to mimic structures involvedin the contact region between gp120 and gp41 are presented in McPhee, D.A., Vth International Conference on AIDS, Montreal, Canada, June 1989,Abstract T.C.O. 35, Putative Interaction Site Between HIV gp120 andgp41: Antiviral Action of Synthetic Peptides, p. 521.

Synthetic peptides of 8 to 25 amino acid residues were tested by McPhee(at 50-100 micrograms/ml) for their ability to inhibit HIV replicationin vitro. A region in gp120 (amino acids 99-119) was identified thatinhibited virus replication as demonstrated by lack of increase inreverse transcriptase (RT) activity and delayed syncytial formation.(McPhee, id.).

Specifically, of the peptides tested and reported by McPhee, gp120(105-117) had marked antiviral activity (99% inhibition of RT activityafter 11 days in culture) (FIG. 2A). (McPhee, D. A., Vaccines 89,Putative Contact Region Between HIV Envelope Proteins gp120 and gp41:Antiviral Action of Synthetic Peptide Analogs, September 1989, pp.185-189.)

It should also be noted, however, in the reported preliminary experimentthat the 25-residue synthetic peptide gp120 (105-129) had only 50%inhibition of RT activity after 11 days in culture. (McPhee, id., p.185).

This synthetic inhibitory peptide (105-129) tested by McPhee did notspan entirely the complementary hydrophobic and counterpart gp41 regionthat contains the fusion-associated epitope, as does the syntheticinhibitory peptide gp120 (107-134) described and represented above bythe Applicant.

EXAMPLE 17

Development and Use of Anti-Idiotypic Antibodies

The human monoclonal antibody Anti-gp41 may be injected as an immunogeninto another species, such as mice, in order to raise anti-idiotypicantibodies to the Anti-gp41. Monoclonal anti-idiotypes could also bedeveloped. Anti-idiotypes could be screened for using available assaytechniques effecting a competition between the labeled peptide and thepotential anti-idiotypes. Those anti-idiotypes capable of displacing thepeptide in assay could then be used in place of the peptide for purposesof immunization or diagnosis.

EXAMPLE 18

Neutralizatiom of HIV-1 (SF2) Infectivity by Human Monoclonal AntibodyDirected Against gp41

The free virus neutralization assay is conducted as follows: Aliquots ofseronegative sera or affinity column (Protein G) purified humanmonoclonal anti-gp41 antibody (Clone 3 Antibody) (dilutions 1:10 and1:100) are incubated with HIV-1 (SF2) for 1 hour at 37° C. The virus andantibody sample is then reacted for 3 hours at 37° C. with PHAstimulated (3 days) peripheral blood mononuclear cells (PBMC) obtainedfrom normal donors. The supernatant is removed and replaced by growthmedia after the 3 hour incubation. On day 8 postinfection, thesupernatant media (quadruplicates) are assayed for reverse transcriptase(RT) activity.

A human monoclonal antibody (Clone 3 Antibody) concentration of 100micrograms/ml produced a 96% inhibition of free virus (SF2) infectivityof human mononuclear cells as demonstrated by lack of increase inreverse transcriptase (RT) activity when cell-free supernatant fluidswere analyzed on day 8 postinfection (Table 5).

                  TABLE FIVE                                                      ______________________________________                                        Neutralization of HIV-1 (SF2) Infectivity                                     By Human Monoclonal Antibody                                                  (Clone 3 Antibody)                                                            Directed Against gp41                                                                Reverse Transcriptase                                                         (RT) Activity (×10.sup.3 cpm/ml)                                                         Human                                                 HIV-1    Normal Patient Serum                                                                         Monoclonal Antibody                                   Virus Strain                                                                           (control)      (10 μg/ml)                                                                           (100 μg/ml)                              ______________________________________                                        SF2      60.0           12.0      2.5                                         Percent  --             80%       96%                                         Inhibition                                                                    ______________________________________                                         Reference: C. ChengMayer, et al., Proceedings of the National Academy of      Sciences, USA, Volume 85, pp. 2815-2819, April 1988, Identification of        Human Immunodeficiency Virus Subtypes with Distinct Patterns of               Sensitivity to Serum Neutralization.                                     

EXAMPLE 19

Immunofluorescence Assay by Flow Cytometry

The indirect immunofluorescence staining procedure was performed asdescribed below.

Aliquots of live Sup-T1 cells (10⁶ in 50 microliters PBS with 0.1% BSAand 0.02% sodium azide), either HTLV-IIIB.9 infected or uninfected, wereincubated in parallel procedures with 5 microliters of human monoclonalantibody (Clone 3 Antibody) supernatant for 30 minutes at 4° C. Thecells were washed and resuspended in 25 microliters of a 1:40 dilutionof affinity-purified fluorescein isothiocyanate (FITC)-conjugated(Fab')₂ goat anti-human IgG (gamma chain specific) (Tago, Burlingame,Calif.) for an additional 30 minutes at 4° C. After incubation, thecells were washed repeatedly, fixed in 4% paraformaldehyde, and then tenthousand stained cells were analyzed by flow cytometry using a BectonDickinson FACS analyzer interfaced to a BD Consort 30 (Becton Dickinson,Mountain View, Calif.). Cells uninfected by HTLV-IIIB.9 served as anegative control.

The fluorescence profiles of the binding of human monoclonal antibody(Clone 3 Antibody) to [A] HTLV-IIIB.9 infected Sup-T1 cells (meanrelative fluorescence intensity=127) and [B]uninfected Sup-T1 cells(mean relative fluorescence intensity=7) as measured bycytofluorographic analysis, are represented in FIG. 9, wherein relativelog green fluorescence intensity (abscissa) is plotted versus relativecell number (ordinate).

The human monoclonal antibody (Clone 3 Antibody) reacted in indirectimmunofluorescence assays with a significant proportion of viableHIV-infected cells suggesting that the determinant (epitope) recognizedby the antibody is expressed on the surface of HIV-infected cells (andnot on uninfected cells) and may be an exposed component of the envelopeof HIV.

Therefore, the HIV-specific human monoclonal antibody (Clone 3 Antibody)directed against the transmembrane (TM) envelope gp41 fusion-associatedoctapeptide epitope with the amino acid sequence GCSGKLIC (SEQ NO:1)(602-609^(Wain-Hobson)) demonstrated in cytofluorographic analysis,reactivity to the native TM envelope glycoprotein gp41.

These data suggest that the octapeptide with the amino acid sequenceGCSGKLIC (SEQ NO:1) (602-609^(Wain-Hobson)) represents a nativeantigenic determinant (epitope) of HIV-1 TM envelope glycoprotein gp41which is expressed on the surface of HIV-infected cells and also elicitsa postinfection immune response with the production of neutralizingantibody, e.g., Clone 3 Antibody.

Although preferred embodiments of the invention have been described inthe foregoing Detailed Description, it will be understood that theinvention is not limited to the embodiments disclosed, but is capable ofnumerous rearrangements and modifications without departing from thespirit of the invention. The present invention is therefore intended toencompass such rearrangements and modifications as fall within the scopeand spirit of the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 42                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                    (ix) FEATURE:                                                                (A) NAME/KEY: Domain                                                          (B) LOCATION: 1..8                                                            (D) OTHER INFORMATION: /label=Epitope                                         /note="A neutralizable epitope of gp41 of HIV-1,                              as identified using monoclonal ATCC CRL 10198, is                             contained within this sequence or a portion thereof.                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlyCysSerGlyLysLeuIleCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       LeuGlyIleTrpGlyCy sSerGlyLysLeuIleCys                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LeuAsnAlaTrpGlyCysAlaPheArgGlnValCys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuAsnSerTrpGlyCysAlaPheArgGlnValCys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 17 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlyCysSerGlyLysLeuIleCysThrThrAlaValProTrpAsnAla                              1 51015                                                                       Ser                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaValGluArgTyrLeuLysAspGlnGlnLeuLeuGlyIleTrp                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    ( B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuLysAspGlnGlnLeuLeuGlyIleTrpGlyCysSerGlyLys                                 1510 15                                                                       (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       LeuLeuGlyIleTrpGlyCys SerGlyLysLeuIleCysThrThr                                151015                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyCysSerGlyLysLeuIleCysThrThrAlaValProTrpAsn                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i ) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LeuIleCysThrThrAlaValProTrpAsnAlaSerTrpSerAsn                                 1 51015                                                                       (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AlaValProTrpAsnAlaSerTrpSerAsnLysSerLeuGluGln                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CysSerGlyLysLeuIleCysThrThr                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      IleLysGlnLeuGlnAlaArgIleLeuAlaValGluArgTyrLeuLys                              15 1015                                                                       AspGlnGlnLeuLeuGlyIleTrpGly                                                   2025                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyIleTrpGlyCysSerGlyLysLeuIleCysThrThrAlaValPro                              151015                                                                        TrpAsnAlaSer                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       AspIleIleSerLeuTrpAspGlnSerLeuLysProCysValLysLeu                             151015                                                                        ThrProLeuCysValSerLeuLysCysThrAspLeu                                           2025                                                                         (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      HisGluAspIleIle SerLeuTrpAspGlnSerLeuLysProCysVal                             151015                                                                        LysLeuThrProLeuCysValSerLeuLysCysThrAspLeu                                    20 2530                                                                       (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlnAlaAr gIleLeuAlaValGluArgTyrLeuLysAspGlnGlnLeu                             151015                                                                        LeuGlyIleTrpGlyCysSerGlyLysLeuIleCysThrThr                                     202530                                                                       (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      G lyCysSerGlyLysLeuIleCysThr                                                  15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CysSerGlyLysLeuIleCysThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                     CysSerGlyLysLeuIleCys                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GlyCysSerGlyLysLeuIleCysThrThr                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AlaIleGluLysTyrLeuGluAspGlnAlaGlnLeuAsnAlaTrpGly                              151015                                                                         CysAlaPheArgGlnValCys                                                        20                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      AlaIleGluLysTyrLeuGlnAspGlnAlaArgLeuAsnSerTrpGly                              151015                                                                        CysAlaPheArgGlnValCys                                                         20                                                                             (2) INFORMATION FOR SEQ ID NO:24:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AlaValGluArgTyrLeuLysAspGlnGlnLeuLeuGl yIleTrpGly                             151015                                                                        CysSerGlyLysLeuIleCys                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GlnLeuThrValTrpGlyIleLysGlnLeuGlnAlaArgIleLeu                                 1510 15                                                                       (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyIleLysGlnLeuGlnAlaAr gIleLeuAlaValGluArgTyr                                151015                                                                        (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GlnAlaArgIleLeuAlaValGluArgTyrLeuLysAspGlnGln                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      LeuGlyLeuTrpGlyCysSerGlyLysLeuIleCys                                          15 10                                                                         (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      LeuGlyIleTrpGlyCysS erGlyLysLeuIleCys                                         1510                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                     GlyCysSerGlyLysLeuIleCys                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                         (v) FRAGMENT TYPE: internal                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CysSerGlyLysLeuIleCysThrThr                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (i ii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      SerGlyLysLeuIleCysThrThr                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GlyLysLeuIleCysThrThr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      LysLeuIleCysThrThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      LeuIleCysThrThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                  (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      SerGlyLysLeuIleCysThr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GlyLysLeuIleCysThr                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      LysLeuIleCysThr                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      LeuIleCysThr                                                                  (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (i ii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      SerGlyLysLeuIleCys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                    (iii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      GlyLysLeuIleCys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (i ii) HYPOTHETICAL: NO                                                       (v) FRAGMENT TYPE: internal                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      LysLeuIleCys                                                                  1                                                                         

I claim:
 1. A hybridoma having all of the identifying characteristics ofthe hybridoma designated Clone 3 having A.T.C.C. Accession No. CRL10198, said hybridoma producing a human monoclonal antibody whichimmunologically binds to the transmembrane glycoprotein, gp41, of HumanImmunodeficiency Virus Type
 1. 2. A human monoclonal antibody producedby the hybridoma of claim 1.